Project Summary/Abstract Transcription initiation plays a pivotal role in the establishment and regulation of cellular phenotypes in all organisms, yet the molecular mechanism of initiation in eukaryotes remains unclear. The process involves the recruitment of RNA polymerase (RNAP), DNA melting, transcription start-site scanning, the initiation of NTP hydrolysis by RNAP, and the transition into processive transcription elongation. In Eukaryotes, transcription is initiated by the pre-initiation complex (PIC) which minimally consists of of TATA-box binding protein (TBP), TFIIB, IIE, IIF, IIH, and RNAP II. Due to the indisputable biological significance of this complex, a plethora of biochemical and structural data have been collected on PICs with the goal of understanding mechanism. These studies have identified and described PIC components and have provided illuminating images of the complex that are exceptionally useful for developing mechanistic hypotheses. However, as structures are snapshots, they represent static images and lack information regarding the dynamics of the PIC factors and the DNA template during initiation. Ultimately, knowledge of how the complex works requires understanding how the PIC transitions between defined structural states along the pathway to initiation. We have begun to successfully observe the dynamic transitions between intermediate states using single-molecule assays well-suited to following transcription initiation. Our long-term goal is to determine the mechanisms behind transcription initiation and its regulation by promoter sequence, transcription factors, and transcriptional activators. This proposal aims to determine the structural transitions of the DNA template induced by PIC activity by applying single-molecule biophysical assays to purified transcription factors from both Saccharomyces cerevisiae and Homo sapiens. The combination of our magnetic tweezers and optical tweezers studies will allow us to make distinct insights into a complex and unsynchronized process. The use of both yeast and human systems will uniquely allow us to compare initiation mechanisms between these highly homologous complexes that exhibit distinct activities in relation to their propensity to scan for start-sites and their ability to be activated by superhelical DNA. We will test competing models for the mechanism of start-site scanning, directly measure the rate and processivity of the recently demonstrated dsDNA translocase activity of Ssl2 in the context of TFIIH, and will test the hypothesis that the 5 bp open complex we have observed in preliminary work on yeast PICs is a conserved feature of eukaryotic initiation by monitoring DNA opening by human PICs. The proposed research is innovative because it uses high-resolution single-molecule techniques to directly measure PIC-dependent conformational changes of promoter DNA and the dsDNA translocation activity of TFIIH in real-time. The results from the project will have a large impact as they will answer fundamental mechanistic questions regarding the dynamic processes that lead to transcription initiation in Eukaryotes.